Mutations play critical roles in cancer initiation and genetic disorders, as well as in microbial pathogenesis and resistance to drugs. Because mutations also generate genetic variability that is required for evolution, cells have mechanisms not only for avoiding excess mutation during normal growth conditions, but also for promoting mutations in response to environmental and physiological stress. In this proposal we will investigate of a recently discovered pathway for mutagenesis that was found to be triggered, surprisingly, by a mutation (mutA) in a gene coding for a tRNA. Our investigation in the previous funding period has led to several important discoveries: mistranslation induced by genetic defects in protein synthesis, or exposure to the antibiotic streptomycin, can induce this mutagenic pathway, named TSM for translational stress-induced mutagenesis. TSM is distinct from the well known SOS mutagenesis pathway, and appears to require homologous recombination functions under certain conditions. TSM appears to be mediated by error-prone DMA synthesis by a modified form of DMA polymerase III. These findings imply the existence of previously undescribed links between translation and DMA replication, and raise the possibility that mutator phenotypes with significance for cell survival, adaptation, cancer initiation, and aging can arise through defects in a much larger repertoire of target genes than previously recognized. The induction of this pathway by antibiotic exposure, and potentially by amino acid starvation suggest that environmental and physiological stress can also increase mutagenesis by this pathway. Our work further shows that aminoglycoside antibiotics pose a mutagenic risk through this previously unrecognized mechanism. Here, we propose to investigate the mechanisms by which mistranslation leads this mutator phenotype through the following three Specific Aims. (1) Test the hypothesis that direct mistranslation of a replicative DMA polymerase leads to the TSM phenotype. (2) Test the hypothesis that in Rec- mutA cells there is selective killing of cells undergoing episodic aberrant replication cycles. (3) Test the hypothesis that the TSM phenotype results from episodic hyper-mutagenesis cycles.